Adenovirus (Ad) DNA replication will be studied in infected nuclear extracts which have been shown to elongate viral DNA by normal intracellular mechanisms. This system has been used to study a temperature sensitive mutant (H5ts125) which is defective in a DNA binding protein (DBP) necessary for Ad DNA synthesis both in whole cells and in nuclear extracts. These DNA replication extracts are dependent on exogenously added DBP. The effects of post-translational modifications such as phosphorylation and polypeptide cleavage of the DBP will be assessed in these complementation assays. The enzymology of Ad DNA replication can be studied in these in vitro systems. We are purifying DNA polymerases bound to Ad replication complexes and comparing the details of structure and function of these bound polymerases with those that are either found free in the infected cell or in unifected cells. Attempts to reconstitute replication complexes that can synthesize full size Ad DNA products will be made using purified viral DNAs and various protein fractions. The requirements for specific DNA polymerases as well as other proteins will be examined in the reconstituted replication complexes. Several selective polymerase inhibitors are available to help compare the requirements for viral DNA replication in whole cells with those in our in vitro systems. Aphidicolin, which we have shown to be an inhibitor of mammalian cell DNA polymerase alpha, will be used. Dideoxythymidine, an inhibitor of gamma polymerase, is also available for these studies. Using this viral model system, we will be able to explore the details of DNA replication in mammalian cells. Some of the differences between virus and cellular pathways may provide clues to the oncogenic potential of the adenoviruses or to ways that inhibition of adenoviral growth may be achieved by selective inhibition of DNA synthesis.